Modulators of Prostate-Specific G-Protein Receptor (PSGR/OR51E2) and Methods of Using Same

ABSTRACT

The present disclosure provides, in part, modulators of prostate-specific G-protein receptor (OR51E2/PSGR) and methods of treating, preventing, and diagnosing prostate cancer using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/415,591, filed Nov. 1, 2016, the disclosure of which is herebyincorporated by cross-reference in its entirety.

FEDERAL FUNDING LEGEND

The work described herein was funded, in whole or in part, using fundsfrom the Federal Government under NIH Grant No.: R01 DC014423.Consequently, the Federal Government has certain rights to thisinvention.

BACKGROUND

Prostate cancer is the second most common cancer in men, accounting for70% of all cancer cases in the developed world. Most deaths fromprostate cancer are due to the progression of localized disease intometastatic, castration-resistant prostate cancer (CRPC). Androgendeprivation therapy is an established treatment for advanced prostatecancer. However, many men eventually fail this therapy and die of CRPC.Recent research attributes the progression of CRPC to neuroendocrinetrans-differentiation (NEtD) of cancerous prostate cells. However, themechanism through which NEtD occurs in prostate cancer remains unclear.Clinical observations have suggested that NEtD correlates with cancerprogression and poor prognosis.

The presence of olfactory receptor OR51E2, also known as ProstateSpecific G-protein Receptor (PSGR), in prostate cancer is welldocumented, but its function is not completely understood. This Gprotein-coupled receptor (GPCR) is expressed in healthy prostate tissueand is significantly over-expressed in prostate cancer. Furthermore,increased expression of OR51E2/PSGR in CRPC has been documented, but itsrole and function in disease progression is currently unknown. Xu et al.demonstrated an increased abundance of OR51E2 in LNCaP cells duringandrogen deprivation. Androgen deprivation results in G0-G1 arrest,cellular senescence, an NE cell-like phenotype, and development ofhighly aggressive clones. (Xu et al. (2000) Cancer Research 60:6568-72).

Furthermore, epidemiological, histopathological, and genetic studieshave shown that chronic infection and inflammation are important inprostate carcinogenesis. Many agents can induce inflammation of theprostate. One such agent is Propionibacterium acnes, which ispredominantly a skin commensal bacterium, but is also found in the oralcavity and gastrointestinal tract. P. acnes is involved in thepathogenesis of acne and is often recognized as an opportunisticpathogen and the cause of chronic post-operative prosthetic jointinfections, osteomyelitis, and endocarditis. P. acnes infection inducesa strong inflammatory response and IL-6, IL-8, and GM-CSF secretion. P.acnes produce propionic acid (PA), which causes acute and chronicprostatitis. Chronic infection produces a constant supply of PA, whichacts as an agonist for OR51E2. Short-term activation of OR51E2 inprostate epithelial cells (RWPE-2) causes an inflammatory reaction,while prolonged/chronic activation may facilitate NEtD, thuscontributing to a more aggressive phenotype.

Currently identified agonists for OR51E2 include propionic acid, aceticacid, androstenone derivatives, and beta-ionone. The only knownantagonist is alpha-ionone, an aroma compound. However, it is unknownwhether any of these ligands have an active role in prostate cancerpathogenesis.

The incidence of prostate cancer is increasing worldwide and there is anurgent need for better diagnostic strategies to distinguish betweenindolent and aggressive tumors, and to develop more efficacioustreatment options for highly aggressive tumors. The results describedherein demonstrate that chronic agonist-mediated activation of theOR51E2/PSGR receptor can turn this receptor into an oncogene and therebyfacilitate cellular progression and transformation resulting in NEtD, acharacteristic phenotype of CRCP. Additionally, the ligands describedherein represent potential novel anti-cancer and diagnostic agents.Results from the studies of the present invention assist in defining therole and function of OR51E2/PSGR. In particular, the identification ofnovel metabolite-agonists provides important molecular and biochemicalinsights into the biological role of this receptor in prostate tissuephysiology and pathophysiology. Finally, the inducement of NEtD byprolonged activation of OR51E2 by PA, a product of P. acnesfermentation, elucidates a causal link between chronic inflammation andNED in prostate cancer.

BRIEF SUMMARY OF THE INVENTION

The present disclosure is based, in part, on the finding that modulationof the OR51E2 receptor results in significant phenotypic changesindicative of neuroendocrine phenotypes, which are resistant to currenttreatments. These results demonstrate that the OR51E2/PSGR receptor is avalid therapeutic target for treating, preventing, and diagnosingcastrate-resistant prostate cancer (CRPC).

One aspect of the present disclosure provides a method of treatingprostate cancer or preventing the progression of prostate cancer in asubject in need thereof, comprising: administering to a subject atherapeutically effective amount of one or more OR51E2 ligands, whereinthe ligand binds to OR51E2 on a prostate cancer cell and impedes theprogression of the prostate cancer cell.

In some embodiments of the above aspect of the invention, the OR51E2ligand is an OR51E2 agonist and/or an OR51E2 antagonist. In otherembodiments, the OR51E2 ligand is estriol, epitestosterone, 19-OH AD(19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione,D-alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK(N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvicacid, adenosine 2′,3′-cyclic phosphate, gamma-CEHC, tetrahydrocurcumin,N-acetylglutamic acid, L-histidinol, bradykinin, 8-Hydroxyguanine,imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid,glycine, propionic acid, or 13-cis retinoic acid.

In some embodiments of the above aspects of the invention, the prostatecancer cell is a castrate resistant prostate cancer cell.

In some embodiments of the above aspects of the invention, the subjectsuffers from chronic infection or inflammation. In other embodiments,the subject suffers from a P. acnes infection.

Another aspect of the present disclosure provides a method of impedingthe progression of a prostate cancer cell, comprising contacting theprostate cancer cell with one or more OR51E2 ligands.

In some embodiments of the above aspects of the invention, the OR51E2ligand is an OR51E2 agonist and/or an OR51E2 antagonist. In otherembodiments, the OR51E2 ligand is estriol, epitestosterone, 19-OH AD(19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione,D-Alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK(N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvicacid, adenosine 2′,3′-cyclic phosphate, gamma-CEHC, tetrahydrocurcumin,N-acetylglutamic acid, L-histidinol, bradykinin, 8-hydroxyguanine,imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid,glycine, propionic acid, or 13-cis retinoic acid.

In some embodiments of the above aspects of the invention, the prostatecancer cell is a castrate resistant prostate cancer cell.

Yet another aspect of the present disclosure provides a method ofdiagnosing prostate cancer in a subject, comprising: a) obtaining abiological sample from the subject; b) contacting the sample with one ormore OR51E2 ligands; c) detecting an increase and/or decrease in thelevel of one or more metabolites associated with ligand-bound OR51E2 inthe sample as compared to a sample not contacted with one or more OR51E2ligands; and d) identifying the presence of prostate cancer based on theincrease and/or decrease of said metabolites.

In some embodiments of the above aspect of the invention, the method ofdiagnosing prostate cancer in a subject comprises detecting a decreasein the level of lactic acid, serine, threonine, glucose-6 phosphate,fructose-6 phosphate, fumaric acid, glutamic acid, beta-alanine,ornithine, and inosine. In other embodiments, the method of diagnosingprostate cancer in a subject further comprises detecting an increase inthe level of intracellular phosphoenolpyruvate and an increase in thelevel of extracellular levels of cystine, aparagine, glutaric acid,guanine, and glutamine.

In some embodiments of the above aspects of the invention, the OR51E2ligand is an OR51E2 agonist and/or an OR51E2 antagonist. In otherembodiments, the OR51E2 ligand is estriol, epitestosterone, 19-OH AD(19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione,D-Alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK(N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvicacid, adenosine 2′,3′-cyclic phosphate, gamma-CEHC, tetrahydrocurcumin,N-acetylglutamic acid, L-histidinol, bradykinin, 8-hydroxyguanine,imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid,glycine, propionic acid, or 13-cis retinoic acid.

In some embodiments of the above aspects of the invention, the presenceof NEtD of prostate cancer cells is identified.

In some embodiments of the above aspects of the invention, thebiological sample is a tissue sample, blood sample, serum sample, orurine sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and other features of the invention are explainedin the following description, taken in connection with the accompanyingdrawings, wherein:

FIG. 1 is a schematic showing prostate epithelial cell differentiationto the NED phenotype.

FIG. 2 is a schematic showing the odorant receptor (OR) and neighboringtransmembrane proteins involved in the signaling in the olfactoryneurons.

FIG. 3 is a homology model of OR51E2.

FIG. 4 is a homology model showing the preferred docking conformation ofthe AFMK ligand.

FIG. 5 is a general schematic of a luciferase assay. (ThermoFisherScientific, Luciferase Reporters, Protein Biology Resource Library,available atwww.thermofisher.com/sa/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/luciferase-reporters.html).

FIG. 6 is a schematic of the in vitro luciferase assay used to test thepotency of OR51E2 ligands (adapted from Zhuang et al. (2008) NatureProtocols 3:1402-13).

FIG. 7 is a dose-response curve showing the effect of propionic acid onOR51E2 receptor activation.

FIG. 8 is a dose-response curve showing the effect of 19-OH AD on OR51E2receptor activation. Responses are normalized to the responses withno-agonist control. Results are mean +/−SEM, n=6.

FIG. 9 is a dose-response curve showing the effect of AFMK on OR51E2receptor activation.

FIG. 10 is a dose-response curve showing the effect of hydroxy pyruvicacid on OR51E2 receptor activation.

FIG. 11 is a dose-response curve showing the effect of kojibiose onOR51E2 receptor activation.

FIG. 12 is a dose-response curve showing the effect of n-acetylglutamicacid on OR51E2 receptor activation.

FIG. 13 is a dose-response curve showing the effect of pelargonidin onOR51E2 receptor activation.

FIG. 14 is a dose-response curve showing the effect of androstanedioneon OR51E2 receptor activation.

FIG. 15 is a dose-response curve showing the effect of estriol on OR51E2receptor activation.

FIG. 16 is a dose-response curve showing the effect of glycyl-glycine onOR51E2 receptor activation.

FIG. 17 is a dose-response curve showing the antagonistic effect of13-cis RA on OR51E2 receptor activation. Responses have been normalizedto the responses with 1 mM PA. Results are mean +/−SEM, n=3.

FIG. 18 is a dose-response curve showing the effect of 19-OHAD on OR51E2receptor activation in the absence (square) and presence (triangle) of13 cis-RA.

FIG. 19 is a schematic showing the production of 19-OH AD by P450aromatase.

FIG. 20 is a schematic of the in vitro assays used to studyneuroendocrine trans-differentiation in prostate cancer cells via theactivation of OR51E2/PSGR with endogenous metabolites.

FIG. 21A is a representative gel image showing transcript levels ofseveral genes after 12 days incubation with 19-OH AD and AFMK, ascompared to control by RT-PCR analysis. OR: OR51E2, NSE: neuron specificenolase, AMACR: alpha-methylacyl-CoA racemase, AR: androgen receptor,GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; K18: keratin K18. FIG.21B is a graph of the transcript levels of markers after stimulationwith agonists for 12 days, N=3 to 6, unpaired t-test, **P<0.01. *P<0.05

FIG. 22A is a graph showing transcript levels of the OR51E2 gene incells treated with 13-cis RA as compared to control (untreated) cells.FIG. 22B is a graph showing transcript levels of the NSE gene in cellstreated with 13-cis RA as compared to control (untreated) cells.

FIG. 23 is a gel showing transcript levels of several genes after 72hours incubation with 13-cis RA as compared to control by RT-PCRanalysis. OR: OR51E2, NSE: neuron specific enolase, AMACR:alpha-methylacyl-CoA racemase, Cav3.2-T-type calcium channel,AR-androgen receptor, GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

FIG. 24A-24F shows heatmaps of the top 15 extracellular andintracellular metabolites identified after stimulation with 19-OH AD,AFMK, and PA for 72 hours. The top 15 extracellular metabolitesidentified after stimulation with 19-OH AD (FIG. 24A), AFMK (FIG. 24B),and PA (FIG. 24C). The top 15 intracellular metabolites identified afterstimulation with 19-OH AD (FIG. 24D), AFMK (FIG. 24E), and PA (FIG.24F). Heatmaps are based on the Pearson correlation analysis (Ward) andindicate annotated metabolites identified by t-test (P<0.05, FDR<0.1,n=6). Columns correspond to the samples treated with agonists (S1-6) andcontrol (S7-12), and rows correspond to annotated metabolites. Controlsamples (n=6 biological replicates, t-test, P<0.05, MetaboAnalyst 3.0software).

FIG. 25A shows a microscope image of prostate cancer cells growing inregular medium have no NED, shown by absence of neurosecretory granules.FIG. 25B shows a microscope image of prostate cancer cells growing inandrogen depleted medium, which show neurosecretory granules, a sign ofNED.

FIG. 26A-26B shows microscope images of the secretory phenotype of LNCaPcells indicative of NED. FIG. 26A shows a microscope image of cellsincubated in regular medium. FIG. 26B shows a microscope image of cellsincubated in androgen-deprived medium (charcoal-dextran treated medium)for 5 days.

FIG. 27 is a graph of a cell viability/proliferation assay of LNCaPcells treated with 13-cis RA for a 7-day incubation period as comparedto control (untreated) cells.

FIG. 28 is a schematic of the assay to study induced NED in prostateepithelial cells via prolonged infection with P. acnes with increasedsecretion of PA.

DETAILED DESCRIPTION OF THE INVENTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to embodiments andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of thedisclosure is thereby intended, such alteration and furthermodifications of the disclosure as illustrated herein, beingcontemplated as would normally occur to one skilled in the art to whichthe disclosure relates.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

Articles “a” and “an” are used herein to refer to one or to more thanone (i.e. at least one) of the grammatical object of the article. By wayof example, “an element” means at least one element and can include morethan one element.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items. As used herein, the singularforms “a,” “an,” and “the” are intended to include the plural forms aswell as the singular forms, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, steps, operations, elements, components, and/or groupsthereof.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by onehaving ordinary skill in the art to which this disclosure belongs. Itwill be further understood that terms, such as those defined in commonlyused dictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art and thepresent disclosure and will not be interpreted in an idealized or overlyformal sense unless expressly so defined herein.

In describing the invention, it will be understood that a number ofaspects and embodiments are disclosed. Each of these has individualbenefit and each can also be used in conjunction with one or more, or insome cases all, of the other disclosed aspects and embodiments.Accordingly, for the sake of clarity, this description will refrain fromrepeating every possible combination of the individual aspects andembodiments in an unnecessary fashion. Nevertheless, the specificationand claims should be read with the understanding that such combinationsare implicitly discloses, and are entirely within the scope of theinvention and the claims, unless otherwise specified.

The present disclosure is based, in part, on the finding that modulationof the olfactory receptor 51E2 (OR51E2), also as known as theProstate-Specific G-Protein Coupled Receptor (PSGR), results insignificant phenotypic changes indicative of neuroendocrine phenotypes,which are resistant to current treatments.

The terms “OR51E2,” “PSGR,” and “OR51E2/PSGR” are used interchangeablyto refer to the olfactory receptor 51E2 (OR51E2) receptor.

Neuroendocrine differentiation: Neuroendocrine (NE) cells are scatteredthrough the epithelium compartment of normal human prostate. NE cellsare typically responsible for growth, differentiation, and secretoryactivity of the prostatic epithelium. As prostate cancer advances,epithelial cells throughout the prostate epithelium differentiate intoneuroendocrine-like (NE-like) cells. NE-like cells contain dendriticcellular extensions and neurosecretory granules containing peptides andneuropeptides, lack androgen receptors, release mitogenic factors andare highly aggressive. Increase in cAMP, induces NEtrans-differentiation (FIG. 1).

Prostate-Specific G-Protein Coupled Receptor (PSGR/OR51E2): Odorantreceptors, also known as olfactory receptors, are G-protein coupledreceptors (GPCRs) found in the brain, skeletal muscle, gastrointestinaltract, sperm, and other tissues. Olfactory receptors share aseven-transmembrane domain receptors, and are responsible for therecognition and G protein-mediated transduction of odorant signals.Olfactory signaling generates increase in cellular cAMP, which createsaction potentials in the olfactory neurons. OR51E2 expression inprostate tissue increases as prostate cancer progresses. As disclosedherein, activation of OR51E2 increases expression of neuroendocrinemarkers, and thus NE-like cells (FIG. 2).

As disclosed herein, OR51E2/PSGR is a therapeutic target for treating,preventing, and diagnosing castrate-resistant prostate cancer (CRPC). Insilico (homology modeling and virtual ligand screening) and in vitro(heterologous cell expression system and luciferase assay) approacheswere used to identify and validate modulators (agonists, antagonists,partial antagonists, and inverse agonists) of OR51E2/PSGR.

As used herein, the term “prostate cancer” refers to cancer that occursin the prostate gland and includes, but is not limited to, benignprostatic hyperplasia (BPH), prostatic adenocarcinoma, small cellcarcinoma, squamous cell carcinoma, prostatic sarcomas, transitionalcell carcinomas, and castrate-resistant prostate cancer (CRPC) (alsoreferred to as androgen independent prostate cancer). Human prostatecancer cell lines used to research prostate cancer include, but are notlimited to, DU145 cells, LNCaP cells and PC-3 cells.

As used herein, the terms “modulator” and “ligand” are usedinterchangeably to refer to an agent that is capable of positively ornegatively impacting basic cellular functions, such as cellproliferation, progression, growth, spread, survival, and/or motility,and is involved in metabolic homeostasis, inflammation, or angiogenicprocesses. Furthermore, a modulator or a ligand is a substance thatforms a complex with a biomolecule to serve a biological purpose. Inprotein-ligand binding, for example, the ligand is usually a moleculethat induces a signal upon binding to a site on a target protein. Thebinding typically results in a change of conformation of the targetprotein. Modulator or ligand binding to a receptor protein, such asOR51E2, alters the chemical conformation by affecting thethree-dimensional shape orientation. The conformation of a receptorprotein composes the functional state. Modulator or ligand binding to areceptor protein, such as OR51E2, can also alter expression patterns orlevels of the receptor.

Modulators and ligands include substrates, inhibitors, activators,neurotransmitters, agonists, antagonists, inverse agonists, inverseantagonists, partial agonists, and partial antagonists. Modulators andligands include, but are not limited to, chemical compounds, such asendogenous metabolites, non-endogenous metabolites, and syntheticchemical compounds, polypeptides, amino acid residues, nucleic acids,siRNA, and antibodies. Examples of modulators and ligands of OR51E2include, but are not limited to, estriol, epitestosterone, 19-OH AD(19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione,D-alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK(N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvicacid, adenosine 2′,3′-cyclic phosphate, gamma-CEHC, tetrahydrocurcumin,N-acetylglutamic acid, L-histidinol, bradykinin, 8-Hydroxyguanine,imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid,glycine, propionic acid, and 13-cis retinoic acid, and isomers thereof.

In certain embodiments, the OR51E2 ligand is 13-cis retinoic acid, andisomers thereof.

Included within the scope of the modulators and ligands of thedisclosure are derivatives of modulators and ligands, such as isotopevariants, substitution variants, and the like, as well as derivativesdesigned to provide for more favorable properties in vitro or in vivo.In a non-limiting example, the modulators and ligands may be covalentlybound to a biologically acceptable polymer.

As used herein, the term “agonist” refers to a modulator or ligand thatbinds to a receptor and activates the receptor to produce a biologicalresponse. OR51E2 agonists can be identified by the in silico and invitro assays described herein. Examples of OR51E2/PSGR agonists include,but are not limited to, estriol, epitestosterone, 19-OH AD(19-hydroxyandrost-4-ene-3,17-dione), palmitic acid, androstenedione,D-Alanyl-d-alanine, glycylglycine, kojibiose, urea, AFMK(N-acetyl-N-formyl-5-metoxykynurenamine), pelargonidin, hydroxypyruvicacid, adenosine 2′,3′-cyclic phosphate, gamma-CEHC, tetrahydrocurcumin,N-acetylglutamic acid, L-histidinol, bradykinin, 8-Hydroxyguanine,imidazolone, 2-pyrrolidinone, 2-ketoglutaric acid, L-glyceric acid,glycine, and propionic acid, and isomers thereof.

As used herein, the term “antagonist” refers to a modulator or ligandthat blocks, impedes, or dampens a biological response by binding to andblocking a receptor rather than activating it. OR51E2 antagonists can beidentified by the in silico and in vitro assays described herein.Examples of OR51E2/PSGR antagonists include, but are not limited to13-cis retinoic acid (13-cis RA), and isomers thereof.

As used herein, the term “inverse agonist” refers to a modulator orligand that binds to the same receptor as an agonist but induces abiological response opposite to that agonist. OR51E2 inverse agonistscan be identified by the in silico and in vitro assays described herein.Examples of OR51E2/PSGR inverse agonists include, but are not limitedto, 13-cis retinoic acid (13-cis RA), and isomers thereof.

As use herein, the term “partial antagonist” refers to a modulator orligand that can bind to a receptor but does not completely block thereceptor's effects, but rather decreases the maximum potential of thereceptor. OR51E2 partial antagonists can be identified by the in silicoand in vitro assays described herein. Examples of OR51E2/PSGR partialantagonists include, but are not limited to, and 13-cis retinoic acid(13-cis RA), and isomers thereof.

Identification of PSGR/OR51E2 modulators and their effect on prostatecancer cell phenotype can be investigated using cellviability/proliferation assays and by analyzing metabolomics signaturesof neuroendocrine differentiation.

Chronic agonist-mediated activation of the OR51E2/PSGR receptor can turnthis receptor into an oncogene/oncoprotein, and thereby facilitatecellular transformation resulting in neuroendocrinetrans-differentiation (NEtD), a characteristic phenotype of castrateresistant prostate cancer (CRCP). An oncogene is a gene that has thepotential to cause cancer, and in tumor cells, oncogenes can be mutatedand/or expressed at high levels. Inhibitors or antagonists and inverseagonists of the OR51E2/PSGR receptor may be used as novel therapeuticagents, in order to slow down NEtD progression in the late stage ofCRCP.

One aspect of the present disclosure provides a method of treatingprostate cancer or preventing the progression of prostate cancer in asubject in need thereof, comprising: administering to a subject atherapeutically effective amount of one or more OR51E2 ligands, whereinthe ligand binds to OR51E2 on a prostate cancer cell and impedes theprogression of the prostate cancer cell.

As used herein, the terms “treating” and “treatment” are usedinterchangeably to refer to both therapeutic treatment and prophylacticor preventative measures. It refers to curing, attenuating, alleviating,minimizing, or suppressing the deleterious effects of a disease state,disease progression, disease causative agent (e.g., bacteria orviruses), or other abnormal condition.

As used herein, the terms “preventing” and “prevention” are usedinterchangeably to refer to impeding, delaying, halting, or reversingthe progression of a disease, such as prostate cancer. For example,preventing prostate cancer can mean preventing a prostate cancer cellfrom differentiation into NE-like cells, a characteristic phenotype ofCRCP.

The term “progression” or “tumor progression” as used herein refer tothe growth, development, differentiation, and proliferation of prostatetumor cells at any stage and grade. In some embodiments, progressionrefers to the advancement from normal prostate epithelial cells topre-invasive lesions. In some embodiments, progression refers to theadvancement of indolent tumors to aggressive tumors. In someembodiments, tumor progression can be characterized by increased growthspeed and invasiveness of the prostate tumor cells. As a result ofprogression, phenotypical changes occur and the prostate tumor canbecome more aggressive and acquires greater malignant potential. Inother embodiments, progression refers to the differentiation of prostateepithelial cells into neuroendocrine-like cells (NEtD), a characteristicphenotype of CRCP.

As used herein, the term “therapeutically effective amount” generallyrefers to an amount of an OR51E2 ligand sufficient to affect a desiredbiological response. Such response may be a beneficial result,including, without limitation, amelioration, reduction, prevention, orelimination of symptoms of a disease or disorder. Therefore, the totalamount of each active component of the OR51E2 ligand is sufficient todemonstrate a meaningful benefit in the patient, including, but notlimited to, treatment of prostate cancer. A “therapeutically effectiveamount” may be administered through one or more preventative ortherapeutic administrations. When the term “therapeutically effectiveamount” is used in reference to a single agent, administered alone, theterm refers to that agent alone, or a composition comprising that agentand one or more pharmaceutically acceptable carriers, excipients,adjuvants, or diluents. When applied to a combination, the term refersto combined amounts of the active agents that produce the therapeuticeffect, or composition(s) comprising the agents, whether administered incombination, consecutively, or simultaneously. The exact amount requiredwill vary from subject to subject, depending, for example, on thespecies, age, and general condition of the subject; the severity of thecondition being treated; and the mode of administration, among otherfactors known and understood by one of ordinary skill in the art. Anappropriate “effective” amount in any individual case may be determinedby one of ordinary skill in the art. Thus, a “therapeutically effectiveamount” will typically fall in a relatively broad range that can bedetermined through routine trials.

The OR51E2 ligands described herein may be administered by any suitableroute of administration. In certain embodiments, an OR51E2 ligand isadministered intravenously, subcutaneously, transdermally,intradermally, intramuscularly, orally, transcutaneously, orintraperitoneally (IP). The OR51E2 ligands may be administered as acomposition comprising the ligand and one or more pharmaceuticallyacceptable carriers, excipients, adjuvants, or diluents.

As used herein, the terms “patient,” “individual,” or “subject” are usedinterchangeably and are intended to include human and non-human animals.Exemplary human subjects include a human patient suffering from prostatecancer, and CRCP in particular. Exemplary human patients may also besuffering from chronic or acute infections or inflammation, such aschronic post-operative prosthetic joint infections, osteomyelitis,endocarditis, and chronic prostatitis, caused by an infection by abacterium, such as P. acnes. The term “non-human animals” includes allvertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles)and mammals, such as non-human primates, domesticated and/oragriculturally useful animals (such as sheep, dogs, cats, rabbits, cows,pigs, etc.), and rodents (such as mice, rats, hamsters, guinea pigs,etc.).

In some embodiments, the subject suffers from chronic infection orchronic inflammation.

Chronic infection occurs when the immune system is unable to respond tothe infective agent or pathogen. Chronic infection can be caused byviral, bacterial, or fungal infections. Chronic infections can occur fora variety of reasons, for example, the pathogen might find a way to hideitself within the body. Examples of chronic infections include, but arenot limited to, chronic fatigue syndrome, Epstein barr virus,mycoplasma, HIV, hepatitis, herpes, chronic post-operative jointinfections, such as osteomyelitis and endocarditis, prostatitis, bladderinfections, chlamydia, and urinary tract infections. In certainembodiments, the subject suffers from a chronic infection caused by P.acnes.

Chronic inflammation can be long-term inflammation and can last forseveral months or years. Chronic inflammation can result from a failureto eliminate the cause of an acute inflammation, an autoimmune responseto a self-antigen, or exposure to a low level of a particular irritant,such as a chemical, over a long period of time. Examples of chronicinflammation include, but are not limited to, asthma, chronic pepticulcer, tuberculosis, rheumatoid arthritis, chronic periodontitis,ulcerative colitis, Crohn's disease, chronic sinusitis, and chronicactive hepatitis.

Another aspect of the present disclosure provides a method of impedingthe progression of a prostate cancer cell, comprising contacting theprostate cancer cell with one or more OR51E2 ligands.

Yet another aspect of the present disclosure provides a method ofdiagnosing prostate cancer in a subject, comprising: a) obtaining abiological sample from the subject; b) contacting the sample with one ormore OR51E2 ligands; c) detecting an increase and/or decrease in thelevel of one or more metabolites associated with ligand-bound OR51E2 inthe sample as compared to a sample not contacted with one or more OR51E2ligands; and d) identifying the presence of prostate cancer based on theincrease or decrease of said metabolites.

The biological sample may be any component extracted from the subject,including, but not limited to, blood, serum, plasma, urine, and tissue.

Metabolites associated with ligand-bound OR51E2 include, but are notlimited to, 2-deoxyglucose, 2-ketoleucine, 3-phosphoglyceric,adenosine/inosine, alanine, alpha ketoglutaric, asparagine, asparticacid, benzoic acid, beta-alanine, cystine, dehydroalanine, ethanolamine,fructose-6-phosphate, fumaric acid, glucose-6-phosphate, glutamic acid,glutamine, glutaric acid, glyceric acid, glycine, guanine, hydroquinone,hydroxyprolines, inosine, lactic acid, lactose, lysine, malic acid,N-acetylaspartic, oleic acid, O-methylphosphate, ornithine, pantothenicacid, pentonic acids, phenylalanine, phosphoenolpyruvate, serine,spermidine, spermine, succinic acid, threitol/erythritol, threonine,threose/erythrose, tyramine, tyrosine, urea, uric acid, and xanthine.

Metabolomics signatures or metabolite profiles of prostate cancer cellsinclude, but are not limited to, lactic acid, serine, threonine,glucose-6 phosphate, fructose-6 phosphate, fumaric acid, glutamic acid,beta-alanine, ornithine, or inosine. In some embodiments, the presenceof NEtD of prostate cancer cells can be indicated by a decrease in theintracellular level of the above-mentioned metabolites, and an increasein intracellular phosphoenolpyruvate, and increase in extracellularlevels of cystine, aparagine, glutaric acid, guanine, glutaminefollowing treatment with an agonist as compared to prostate cancer cellsthat were untreated.

As used herein, the term “diagnose” refers to identifying the nature ofa medical condition of a subject, such as prostate cancer, includingcastrate resistant prostate cancer, from its signs and symptoms.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1: IDENTIFICATION OF BIOLOGICALLY RELEVANT OR51E2/PSGRMETABOLITE-LIGANDS IN SILICO

To discover new, biologically relevant ligands for OR51E2/PSGR, ahomology modeling and an in silico ligand screening approach of ametabolite library was applied. The Modeller v.9.14 program was used toproduce a model of OR51E2 based on the crystal structure of the β2adrenergic receptor. Twenty models were made using an automodel scriptand each model was assessed with DOPE score. The best model was used forvirtual ligand screening (VLS) with ICM Software (MolSoft v.3.8, LLC).Homology model of OR51E2 is shown in FIG. 3.

A library of 2,511 human metabolites were selected from the HumanMetabolome Database (www.HMDB.ca) and virtually screened against ahomology model of OR51E2/PSGR. The VLS result lists metabolitesaccording to their scores, which represent the predicted binding of theligand to the receptor. Results from the VLS, for example, showed thepreferred docking conformation of acetyl-2-formyl-5-methoxykynurenamine(AFMK) ligand bound to OR51E2 (FIG. 4).

The identification of agonists and antagonists, selected from a libraryof human metabolites as opposed to virtually designed compounds, willunravel specific pathways that can be modulated to reverse NEtD ofcancer cells.

EXAMPLE 2: EFFECT OF BIOLOGICALLY RELEVANT OR51E2/PSGRMETABOLITE-LIGANDS ON METABOLISM IN PROSTATE CANCER CELLS IN VITRO

To determine whether OR51E2/PSGR activation mediates neuroendocrinetrans-differentiation (NEtD), which is characteristic for castrateresistant prostate cancer, CRPC, an in vitro luciferase assay was usedto validate the top ligand candidates identified by the in silico methoddescribed in Example 1.

Luciferase Assay

Hana3A cells (modified human embryonic kidney cells) were cultured in 96well plates in M10PSF medium. LNCaP human prostate cancer cells werecultured in both RPMI medium and androgen deprived RPMI medium. Hana3Acells were transfected with CRE-Luc (CREB-dependent luciferase(Firefly)) and SV40-RL promoter plasmids, and pCI and OR51E2 plasmids.Hana3A cells were then stimulated with ligands that were identified inthe virtual screen process of Example 1. Upon ligand binding, anincrease in cAMP drives the expression of Firefly luciferase. Increasein luminescence signal was measured with a luminometer and Optima DataAnalysis software. The luminescent signal is directly proportional toreceptor activation. A general schematic of a luciferase assay is shownin FIG. 5 and a schematic of the assay used in this Example is shown inFIG. 6.

The top 56 compounds from the VLS list were tested using the in vitroexpression and luciferase assay. The potency of the most promisingagonist and antagonist was determined by calculating their EC₅₀ andIC₅₀, respectively. The biologically relevant concentrations of eachmetabolite were individually determined based on available data in theliterature. If no data were available, compounds were tested in therange of 1 nM-100 μM. Responses were normalized to the response with 1mM propionic acid (PA), a previously identified agonist, and also to aresponse with no-control. The effect of PA on OR51E2 is shown in thedose-response curve of FIG. 7.

Results

Twenty four (24), new potent OR51E2 agonists and one potent OR51E2antagonist were identified. The dose response curves for several OR51E2agonists are presented here in the figures indicated after eachcompound: 19-OHAD (19-hydroxyandrostenedione) (FIG. 8), AFMK(acetyl-2-formyl-5-methoxykynurenamine) (FIG. 9), hydroxypyruvic acid(FIG. 10), kojibiose (FIG. 11), N-acetylglutamic acid (FIG. 12),pelargonidin (FIG. 13), androstanedione (FIG. 14), estriol (FIG. 15),and glycyl-glycine (FIG. 16). The EC₅₀ values and efficacy (whencompared to the 1 mM PA response) for all identified OR51E2 agonists areprovided in Table 1.

TABLE 1 Potency (EC₅₀) and efficacy of OR51E2 Agonists EC₅₀ Max(potency) conc. Agonist Name HMDB CAS [M] used Efficacy EstriolHMDB00153 50-27-1 5.30E−05 10 μM 0.344 Epitestosterone HMDB00628481-30-1 6.90E−10 10 μM 0.477 19-OH AD HMDB03955 510-64-5 1.50E−10 10 μM0.890 (19-hydroxyandrost- 4-ene-3,17-dione) Palmitic acid HMDB0022057-10-3 9.80E−09 1 mM 0.927 Androstanedione HMDB00899 846-46-8 7.90E−10100 μM 0.888 D-Alanyl-d-alanine HMDB03459 923-16-0 1.40E−05 3.16 mM1.505 Glycylglycine HMDB11733 556-50-3 1.10E−05 3.16 mM 1.797 KojibioseHMDB11742 NA 1.00E−06 316 μM 0.790 Urea HMDB00294 57-13-6 2.30E−08 10 mM0.580 AFMK (N-acetyl- HMDB04259 52450-38-1 1.20E−05 3.16 mM 1.483N-formyl-5- metoxykynurenamine) Pelargonidin HMDB03263 134-04-3 4.20E−10100 μM 0.621 Hydroxypyruvic acid HMDB01352 1113-60-6 4.20E−07 316 μM1.100 Adenosine 2′,3′- HMDB11616 634-01-5 2.60E−08 3.16 μM 1.025 cyclicphosphate Gamma-CEHC HMDB01931 178167-77-6 6.40E−09 10 μM 1.286Tetrahydrocurcumin HMDB05789 36062-04-1 5.70E−07 316 μM 0.284N-Acetylglutamic acid HMDB01138 1188-37-0 2.30E−10 10 μM 0.879L-Histidinol HMDB03431 4836-52-6 3.50E−11 100 μM 0.578 BradykininHMDB04246 58-82-2 1.30E−09 100 μM 0.762 8-Hydroxyguanine HMDB020325614-64-2 4.40E−13 100 nM 0.570 Imidazolone* HMDB04363 1192-34-37.60E−12 10 μM 0.678 2-Pyrrolidinone HMDB02039 616-45-5 1.90E−09 100 μM0.525 2-Ketoglutaric acid HMDB00208 328-50-7 5.50E−09 1 mM 0.594L-Glyceric acid HMDB06372 28305-26-2 1.90E−09 1 mM 0.898 GlycineHMDB00123 56-40-6 5.80E−08 1 mM 0.613

Additionally, 13-cis retinoic acid (13-cis RA) was identified as apotent OR51E2 antagonist with an IC₅₀ value of 160 nM. A dose responsecurve for 13-cis RA is shown in FIG. 17.

19-OH AD was also tested in Hana3A cells expressing OR51E2 in theabsence and presence of the 13-cis retinoic acid antagonist, furtherdemonstrating the potency of 13-cis RA as an inhibitor of OR51E2. (FIG.18).

Discussion

19-OH AD was one of the most potent agonist identified from the ligandscreen, as indicated by an EC₅₀ of 1.5e⁻¹⁰ M (FIG. 8 and Table 1). 19-OHAD is produced by the enzyme P450 aromatase (CYP19A1), a highlyup-regulated enzyme in prostate cancer (PCa) (FIG. 19), indicating that19-OH AD is a biologically relevant agonist. Furthermore, 19-OH ADamplifies the effects of the renin-angiotensin system (RAS), a systemthat, when over activated, causes hypertension. All RAS components havebeen identified in the prostate and angiotensin II was shown to have arole in prostate cancer development. The correlation between 19-OH AD,RAS and prostate cancer indicates a relationship between 19-OH AD,activation of OR51E2 and prostate cancer.

The other agonists identified by this assay are also biologicallyrelevant. AFMK, a metabolite of kynurenamine, was previously reported tobe abundantly present in aggressive prostate cancers. AFMK mitigatesdamage to DNA through anti-oxidative effects. AFMK's role in mitigatingthe adverse effects of cancer indicates its involvement in prostatecancer.

Glycyl-glycine has been detected in the plasma of patients with prostatecancer. Glycyl-glycine metabolism occurs in digestion and producesglycine. A derivative of glycine is sarcosine, a biomarker for prostatecancer. Further research could support the role of glycyl-glycineactivation of OR51E2 in prostate cancer development.

Kojibiose has been detected in the plasma of patients with prostatecancer. Its prevalence in prostate cancer may point to a potentialrelationship between kojibiose activation of OR51E2 and prostate cancerdevelopment.

Furthermore, the results indicate that 13-cis RA can act via the OR51E2receptor. Isotretinoin, or 13-cis RA, is a potent oral retinoid used forthe treatment of severe acne, and is effective against P. acnesbacterium. 13-cis RA could be an inhibitor of OR51E2 to treat prostatecancer. Previously, Dahiya et al. investigated effects of 13-cis RA onLNCaP cells and found it inhibits cell growth and decreases tumorigenicpotential. (Dahiya et al. (1994) Int. J. Cancer 59(1):126-132).

In addition, propionic acid effects on prostate cancer cells will bestudied, as it was previously identified as agonist for PSGR/OR51E2.Alpha-ionone, an antagonist, will also be tested.

The biologically relevant OR51E2 ligands identified by this in vitroassay can be used as therapeutic and diagnostic agents for prostatecancer. Additionally, this assay is a useful in vitro model to studyneuroendocrine trans-differentiation of prostate cancer cells.

EXAMPLE 3: EFFECT OF BIOLOGICALLY RELEVANT OR51E2/PSGRMETABOLITE-LIGANDS ON EXPRESSION SIGNATURES IN PROSTATE CANCER CELLS INVITRO

To further determine whether OR51E2/PSGR activation mediatesneuroendocrine trans-differentiation (NEtD), prostate cancer cells weretreated with selected metabolite-ligands and their metabolomicssignatures, expression of NE-markers, and viability and proliferationwere analyzed. (FIG. 20).

In Vitro Expression and Metabolomics Signature Assays

To assess the differentiation status of LNCaP cells following treatmentwith an OR51E2 ligand, cells were exposed to the ligands for three days.Biological markers of NED were analyzed using an RT-PCR assay. Thefollowing markers were tested: neuron specific enolase (NSE) andα-methylacyl-CoA racemase (AMACR), an enzyme essential for isomerizationof branched-chained fatty acids that is present at low levels in healthyprostate cells and increased in PCa and in NE-like cells. Furthermore,changes in the expression levels of OR51E2 and androgen receptor (AR)were determined. This experiment will also be conducted using RWPE-2cells.

Expression of keratins K5, K8, and K18 will also be assessed, as RWPE-2cells are positive for K8 and K18. LNCaP cells are positive for K18, andNE cells express K5.

Metabolomics signatures were assessed using untargeted gaschromatography/mass spectrometry (GC/MS) to analyze LNCaP cells treatedwith an OR51E2 ligand for 3 days. There were six biological replicatesfor each treatment and the results were analyzed using MetaboAnalystsoftware. The same analysis will also be conducted using RWPE-2 cells.

The NEtD status was assessed through analysis of viability andproliferation of LNCap cells using use CellTiter-Glo Luminescent cellviability assay (Promega). Reduced proliferation with agonist treatmentwill occur because NE-like cells do not proliferate. Viability andproliferation assays will also be conducted with OR51E2 ligands inRWPE-2 cells.

The OR51E2 gene will be knocked down using an siRNA interferenceapproach in order to confirm that the observed effects are indeedreceptor-specific.

Results

The results indicate that LNCaP cells show alterations in transcriptlevels of several genes implicated in NEtD after 12 days incubation withthe indicated OR51E2 ligands (FIG. 21A and FIG. 21B). For example, 19-OHAD agonist treatment of LNCaP cells increased OR51E2 transcript levels.PA agonist-treatment increased NSE and AMACR transcript levels.Likewise, LNCaP cells treated with 10 μM 13-cisRA for 3 days showsignificantly decreased OR51E2 transcript levels as compared to controlcells (FIG. 22A-22B, FIG. 23). These results are promising and indicatethat OR51E2 activation is associated with NEtD. Further studies usingsiRNA gene knockdown assays, will determine whetheractivated/overexpressed OR51E2 is involved in NEtD.

LNCaP cells incubated with agonists: 100 nM 19-OH AD, 250 μM AFMK, and 1mM PA for 72 hours and analyzed with GC/MS (Agilent 6890N GC-5975-InertMSD) showed significant differences relative to controls (FIG. 24A-24F).GC/MS analysis detected greater than 250 features, and 115 of them wereannotated. In FIG. 24A-24F, the top 15 metabolites are presented showingthe most significant differences in cells treated with agonists whencompared to control cells.

The top 15 extracellular metabolites identified after stimulation with19-OH AD were asparagine, glutaric acid, guanine, cysteine, alphaketoglutaric, 2-ketoleucine, hydroquinone, succinic acid, glyceric acid,n-acetylaspartic, hydroxyprolines, 2-deoxyglucose, beta-alanine, benzoicacid, and urea (FIG. 24A).

The top 15 extracellular metabolites identified after stimulation withAFMK were phenylalanine, serine, lysine, glycine, glutaric acid,cystine, uric acid, tyrosine, xanthine, guanine, glutamine, asparagine,threose/erythrose, alanine, and lactic acid (FIG. 24B).

The top 15 extracellular metabolites identified after stimulation withPA were tyramine, spermine, lactose, guanine, spermidine, glutamine,ornithine, threitol/erythritol, lactic acid, serine, glycine, threonine,dehydroalanine, aspartic acid, and glutamic acid (FIG. 24C).

The top 15 intracellular metabolites identified after stimulation with19-OH AD were tyramine, adenosine/inosine, guanine, alanine, pantothenicacid, fumaric acid, malic acid, serine, threonine, fructose-6-phosphate,beta-alanine, lactic acid, glucose-6-phosphate, tyrosine, and urea (FIG.24D).

The top 15 intracellular metabolites identified after stimulation withAFMK were pentonic acids, spermidine, malic acid, serine, threonine,ethanolamine, oleic acid, inosine, glucose-6-phosphate, lactic acid,ornithine, beta-alanine, fumaric acid, O-methylphosphate, and3-phosphoglyceric (FIG. 24E).

The top 15 intracellular metabolites identified after stimulation withPA were tyramine, spermine, lactose, guanine, spermidine, glutamine,ornithine, threitol/erythritol, lactic acid, serine, glycine, threonine,dehydroalanine, aspartic acid, and glutamic acid (FIG. 24F).

Metabolomics signatures of prostate cancer cells include a decrease inthe level of lactic acid, serine, threonine, glucose-6 phosphate,fructose-6 phosphate, fumaric acid, glutamic acid, beta-alanine,ornithine, and inosine. The presence of NEtD of prostate cancer cellscan be indicated by a decrease in the intracellular level of theabove-mentioned metabolites (FIG. 24D, FIG. 24E and FIG. 24F), and anincrease in intracellular phoshoenol-pyruvate, and an increase inextracellular levels of cystine, aparagine, glutaric acid, guanine,glutamine (FIG. 24A, FIG. 24B and FIG. 24C) following treatment with anagonist as compared to prostate cancer cells that were untreated (FIG.24).

These results also demonstrate that agonist treatment significantlydecreased levels of serine and threonine. Because metabolism of theseamino acids includes one-carbon metabolism, which provides cofactors forbiosynthetic reactions in highly proliferating cells, intracellulardepletion may indicate a general decrease in anabolic reactions inagonist-stimulated cells. Furthermore, lactic acid was also decreased,which is in agreement with the attenuated proliferation ratecharacteristic for NE-like cells. Neuhaus et al. reported thatOR51E2/PSGR activation by beta ionone decreased proliferation of LNCaPcells. (Neuhaus et al. (2009) Journal of Biological Chemistry,284:16218-25).

LNCaP cells incubated in androgen-deprived medium (charcoal-dextrantreated medium) for five days (n=3, two tailed t-test) show thecharacteristic NE-like secretory phenotype as compared to thoseincubated in regulate medium (FIG. 25A-25B and FIG. 26A-26B) and havedecreased levels of serine and threonine, indicating that decreases inthese amino acids may constitute part of the characteristic NEDmetabolic profile (Table 2).

TABLE 2 Significantly decreased metabolites identified in LNCaP celllysates incubated in androgen-deprived medium Metabolite t-test Serine0.00005 Threonine 0.00007 Creatinine 0.0001 Fructose 0.0001 Isoleucine0.001 Myoinositol 0.003 Malic acid 0.007 Glycerol 1- 0.011 phosphateHydroxyprolines 0.015 Glucose 0.016 Aspartic acid 0.017 Citric acid0.023 Valine 0.024 Lactic acid 0.030

Furthermore, a cell viability/proliferation assay of prostate cancerLNCaP cells was performed using 13-cis RA during a 7 day incubationperiod. As shown in FIG. 27, 10 uM 13-cis RA effectively impeded LNCaPgrowth as compared to control (untreated) LNCaP cells.

Discussion

19-OH AD treatment of LNCaP cells increased OR51E2 transcript. PAagonist increased markers of NEtD: NSE and AMACR transcripts. Short termtreatment of LNCaP cells with OR51E2 agonists indicate decreased levelof several amino-acids. The decreased level of lactate inagonist-treated cells is in agreement with decreased glycolysischaracteristic for neuroendocrine cancer phenotype. These resultsindicate that agonist modulation of OR51E2/PSGR shows changes incellular metabolism and phenotypic changes indicative of NED. Theseresults point at the OR51E2/PSGR as a valid therapeutic target indesigning novel treatment strategies for CRPC.

EXAMPLE 4: THE LIGANDS FOR OR51E2/PSGR: A LINK BETWEEN PROSTATICINFLAMMATION AND PROSTATE CANCER

To determine if prolonged infection with P. acnes with increasedsecretion of PA can induce NED in prostate epithelial cells, theexpression of NED markers (chronic infection) and cytokine secretion(acute infection) will be assessed. The ability of the OR51E2 antagonistto modify and reverse NEtD will also be assessed (FIG. 28).

RWPE-2 cells will be infected with P. acnes (ATCC [Manassas, Va.] orNational Collection of Type Cultures [NCTC, Colindale, London, UK])using a slightly modified protocol and with the addition of 15%glycerol. Six biological replicates will be performed for eachexperimental condition. Acute and chronic-prolonged effects will beanalyzed at 24 hours and 12 weeks post-infection. In order to sustainprolonged infection, successive infections will be performed every threeweeks.

RWPE-2 cells will be incubated with 1 mM PA for 24 hours and 12 weeks,and cytokine release (after 24 hours) and metabolomics signatures (after12 weeks) will be measured as described above in Example 3. Cytokinerelease will be measured in RWPE-2 conditioned medium 24 hours postinfection using a 40-plex panel kit ELISA assay (BioRad). Metabolomicssignatures will be analyzed in both cell lysates and conditioned medium12 weeks post-infection using an untargeted metabolomics approach.Statistical and enrichment pathway analysis will be done usingMetaboAnalyst software.

Antagonist-treated, P. acnes-infected cells will be analyzed for theirmetabolomics signatures. At three weeks post-infection, antagonist willbe added to the medium and the cells will be incubated for the followingthree weeks. Metabolomics signatures from the treatment and controlgroups will then be compared using the approach described in Example 3.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods described herein are presentlyrepresentative of preferred embodiments, are exemplary, and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention as defined by the scopeof the claims.

1. A method of diagnosing prostate cancer in a subject, the methodcomprising: i) obtaining a biological sample from the subject; ii)contacting the sample with one or more OR51E2 ligands; iii) detecting adecrease in the level of one or more metabolites as compared to a samplenot contacted with the one or more OR51E2 ligands; and iv) identifyingthe presence of prostate cancer based on the change in the level of saidone or more metabolites.
 2. The method of claim 1, wherein the one ormore metabolites are lactic acid, serine, threonine, glucose-6phosphate, fructose-6 phosphate, fumaric acid, glutamic acid,beta-alanine, ornithine, and inosine.
 3. The method of claim 1, whereinthe one or more metabolites are serine, threonine, creatinine, fructose,isoleucine, myoinositol, malic acid, glycerol 1-phosphate,hydroxyprolines, glucose, aspartic acid, citric acid, valine, and lacticacid.
 4. The method of claim 1, wherein the one or more metabolites areserine and threonine.
 5. The method of claim 1, wherein the one or moreOR51E2 ligands comprises an OR51E2 agonist.
 6. The method of claim 1,wherein the one or more OR51E2 ligands comprises an OR51E2 antagonist.7. The method of claim 1, wherein the one or more OR51E2 ligands areestriol, epitestosterone, 19-hydroxyandrost-4-ene-3,17-dione (19-OH AD),palmitic acid, androstenedione, D-Alanyl-d-alanine, glycylglycine,kojibiose, urea, N-acetyl-N-formyl-5-metoxykynurenamine (AFMK),pelargonidin, hydroxypyruvic acid, adenosine 2′,3′-cyclic phosphate,gamma-CEHC, tetrahydrocurcumin, N-acetylglutamic acid, L-histidinol,bradykinin, 8-hydroxyguanine, imidazolone, 2-pyrrolidinone,2-ketoglutaric acid, L-glyceric acid, glycine, propionic acid, 13-cisretinoic acid, isomers thereof, and/or combinations thereof.
 8. Themethod of claim 1, wherein the one or more OR51E2 ligands is an agonistand wherein the one or more metabolites are serine and threonine.
 9. Themethod of claim 1, wherein the one or more OR51E2 ligands is an agonistand wherein the one or more metabolites is lactic acid.
 10. The methodof claim 1, wherein the one or more OR51E2 ligands is 19-OH AD.
 11. Themethod of claim 1, wherein the one or more OR51E2 ligands is propionicacid.
 12. The method of claim 1, wherein the one or more OR51E2 ligandsis 13-cis retinoic acid.
 13. The method of claim 1, the method furthercomprising detecting an increase in the level of the one or moremetabolites.
 14. The method of claim 13, wherein the one or moremetabolites that are increased are intracellular phoshoenol-pyruvate andextracellular cysteine, asparagine, glutamic acid, guanine, andglutamine.
 15. The method of claim 1, wherein the presence ofcastrate-resistant prostate cancer is identified.
 16. The method ofclaim 1, wherein the presence of neuroendocrine trans-differentiation ofprostate cancer cells is identified.
 17. The method of claim 1, whereinthe biological sample is a tissue sample, blood sample, serum sample,plasma sample, or urine sample.
 18. A method of diagnosing prostatecancer in a subject, the method comprising: i) obtaining a biologicalsample from the subject; ii) contacting the sample with one or moreOR51E2 agonists; iii) detecting a decrease in the level of lactic acid,serine, threonine, glucose-6 phosphate, fructose-6 phosphate, fumaricacid, glutamic acid, beta-alanine, ornithine, or inosine and detectingan increase in the level of phosphoenolpyruvate, cysteine, asparagine,glutamic acid, guanine, or glutamine as compared to a sample notcontacted with the one or more OR51E2 agonists; and iv) identifying thepresence of prostate cancer based on the change in the levels of the oneor more metabolites.
 19. The method of claim 18, wherein the one or moreOR51E2 agonists are 19-OH AD, AFMK, hydroxypyruvic acid, kojibiose,N-acetylglutamic acid, pelargonidin, androstenedione, estriol, and/orglycyl-glycine.
 20. The method of claim 18, wherein the biologicalsample is a tissue sample, blood sample, serum sample, plasma sample, orurine sample.